The invention, in the fields of biochemistry and cell and molecular biology, relates to a novel protein tyrosine phosphatase (PTPase or PTP) protein or glycoprotein, termed PTP-S31, the use of such molecule in pharmaceutical preparations, and pharmaceutical compositions comprising PTP-S31 or functional derivatives thereof. This invention is also directed to nucleic acid molecules encoding the PTP-S31 protein or functional derivative, recombinant expression vectors carrying the nucleic acid molecules, cells containing the recombinant expression vectors, methods for production and identification of PTP-S31 or the DNA coding therefor, antibodies specific for PTP-S31, and methods for screening compounds capable of binding to and inhibiting or stimulating protein tyrosine phosphatase enzymatic activity of PTP-S31.
Phosphorylation of proteins is a fundamental mechanism for regulating diverse cellular processes. While the majority of protein phosphorylation occurs at serine and threonine residues, phosphorylation at tyrosine residues is attracting a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established (Hunter et al., Ann. Rev. Biochem. 54:987-930 (1985), Ullrich et al., Cell 61 :203-212 (1990), Nurse, Nature 344:503-508 (1990), Cantley et al., Cell 64:281-302 (1991)).
Biochemical studies have shown that phosphorylation on tyrosine residues of a variety of cellular proteins is a dynamic process involving competing phosphorylation and dephosphorylation reactions. The regulation of protein tyrosine phosphorylation is mediated by the reciprocal actions of protein tyrosine kinases (PTKases or PTKS) and protein tyrosine phosphatases (PTPs). The tyrosine phosphorylation reactions are catalyzed by PTKs. Tyrosine phosphorylated proteins can be specifically is dephosphorylated through the action of PTPS. The level of protein tyrosine phosphorylation of intracellular substances is determined by the balance of PTK and PTP activities. (Hunter, T., Cell 58:1013-1016 (1989)).
2.1. PTKs
The protein tyrosine kinases (PTKs; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112) are a large family of proteins that includes many growth factor receptors and potential oncogenes. (Hanks et al., Science 241:42-52 (1988)). Many PTKs have been linked to initial signals required for induction or the cell cycle (Weaver et al., Mol. and Cell. Biol. 11(9):4415-4422 (1991)). PTKs comprise a discrete family of enzymes having common ancestry with, but major differences from, serine/threonine-specific protein kinases (Hanks et al., supra). The mechanisms leading to changes in activity of PTKs are best understood in the case of receptor-type PTKs having a transmembrane topology (Ullrich et al. (1990) supra). The binding of specific ligands to the extracellular domain of members of receptor-type PTKs is thought to induce their oligomerization leading to an increase in tyrosine kinase activity and activation of the signal transduction pathways (Ullrich et al., (1990) supra). Deregulation of kinase activity through mutation or overexpression is a well-established mechanism for cell transformation (Hunter et al., (1985) supra; Ullrich et al., (1990) supra).
2.2. PTPS
The protein phosphatases are composed of at least two separate and distinct families (Hunter, T.(1989) supra) the protein serine/threonine phosphatases and the protein tyrosine phosphatases (PTPs; protein-tyrosine-phosphate phosphohydrolase, EC 3.13.48)). The PTPs are a family of proteins that have been classified into two subgroups. The first subgroup is made up of the low molecular weight, intracellular enzymes that contain a single conserved catalytic phosphatase domain. All known intracellular type PTPS contain a single conserved catalytic phosphatase domain.; Examples of the first group of PTPs include (1) placental PTP 1B (Charbonneau et al., Proc. Natl. Acad. Sci. USA 86:5252-5256 (1989); Chernoff et al., Proc. Natl. Acad. Sci. USA 87:2735-2789 (1990)), (2) T-cell PTP (Cool et al. Proc. Natl. Acad. Sci. USA 86:5257-5261 (1989)), (3) rat brain PTP (Guan et al., Proc. Natl. Acad. Sci. USA 87:1501-1502 (1990)), (4) neuronal phosphatase (STEP) (Lombroso et al., Proc. Natl. Acad. Sci. USA 88:7242-7246 (1991)), and (5) cytoplasmic phosphatases that contain a region of homology to cytoskeletal proteins (Guet al., Proc. Natl. Acad. Sci. USA 88:5867-57871 (1991); Yang et al., Proc. Natl. Acad. Sci. USA 88:5949-5953 (1991)).
The second subgroup is made up of the high molecular weight, receptor-linked PTPs, termed RPTPs. RPTPs consist of (a) an intracellular catalytic region, (b) a single transmembrane segment, and (c) a putative ligand-binding extracellular domain. The structures and sizes of the putative ligand-binding extracellular xe2x80x9creqeptorxe2x80x9d domains of RPTPs are quite divergent. In contrast, the intracellular catalytic regions of RPTPs are highly homologous. All RPTPs have two tandemly duplicated catalytic phosphatase homology domains, with the prominent exception of an RPTP termed HPTPxcex2, which has only one catalytic phosphatase domain. (Tsai et al., J. Biol. Chem. 266:10534-10543 (1991)).
One example of RPTPs is a family of proteins termed leukocyte common antigens (LCA) (Ralph, S. J., EMBO J. 6:1251-1257 (1987)) which are high molecular weight glycoproteins expressed on the surface of all leukocytes and their hemopoietic progenitors (Thomas, Ann. Rev. Immunol. 7:339-369 (1989)). A remarkable degree of similarity exists in the sequences of LCA from several species (Charbonneau et al., Proc. Natl. Acad. Sci. USA 85:7182-7186 (1988)). LCA has been referred to in the literature by different names, including T200 (Trowbridge et al., Eur. J. Immunol. 6:557-562 (1962)), B220 for the B lymphocyte form (Coffman et al., Nature 289:681-683 (1981)), the mouse allotypic marker Ly-5 (Komuro et al., Immunogenetics 1:452-456 (1975)), and more recently CD45 (Cobbold et al., Leucocyte Typing III, A. J. McMichael et al., eds., pp. 788-803 (1987)).
CD45 appears to play a critical role in T cell activation (reviewed in Weiss A., Ann. Rev. Genet. 25:487-510 (1991)). For example, T-cell clones that were chemically mutagenized and selected for their failure to express CD45 had impaired responses to T-cell receptor stimuli (Weaver et al., supra). These T-cell clones were functionally defective in their responses to signals transmitted through the T cell antigen receptor, including cytolysis of appropriate targets, proliferation, and lymphokine production (Weaver et al., supra). Other studies indicate that the PTP activity of CD45 plays a role in the activation of pp 561ck, a lymphocyte-specific PTK (Mustelin et al., Proc. Natl. Acad. Sci. USA 86:6302-6306 (1989); ostergaard et al., Proc. Natl. Acad. Sci. USA 86:8959-8963 (1989)). These authors hypothesized that the phosphatase activity of CD45 activates pp56kk by dephosphorylation of a C-terminal tyrosine residue, which may, in turn, be related to T-cell activation.
Another other example of an RPTP is the leukocyte common antigen related molecule (LAR), initially identified as a homologue of LCA (Streuli et al., J. Exp. Med. 168:1523-1530 (1988)). Although the intracellular catalytic region of the LAR molecule contains two catalytic phosphatase homology domains (domain I and domain II), mutational analyses suggested that only domain I had catalytic phosphatase activity, whereas domain II was enzymatically inactive (Streuli et al., EMBO J. 9(8):2399-2407 (1990)). Chemically induced LAR mutants having tyrosine at amino acid position 1379 changed to a phenylalanine were temperature-sensitive (Tsai et al., J. Biol. Chem. 266(16):10534-10543 (1991)).
A recently cloned mouse RPTP, designated mRPTPxcexc, was found to have an extracellular domain that shared some structural motifs with LAR. (Gebbink, M. F. B. G. et al., FEBS Lett. 290:123-130 (1991). These authors also cloned a human homologue of RPTPxcexc and localized the gene on human chromosome 18.
Two Drosophila PTPs, termed DLAR and DPTP, have been predicted based on the sequences of cDNA clones (Streuli et al., Proc. Natl. Acad. Sci. USA 86:8698-8702 (1989)). cDNAs coding for another Drosophila RPTP, termed DPTP 99A, have been cloned and characterized (Hariharan et al., Proc. Natl. Acad. Sci. USA 88:11266-11270 (1991)).
Other examples of RPTPs include RPTP-xcex1, xcex2, xcex3, and xcex6 (Krueger et al., EMBO J. 9:3241-3252 (1990), Sap et al. Proc. Natl. Acad. Sci. USA 67;;6112-6116 (1990), Kaplan et al., Proc. Natl. Acad. Sci. USA 87:7000-7004 (1990), Jirik et al., FEBS Lett. 273:239-242 (1990), Mathews et al., Proc. Natl. Acad. sci. USA 87:4444-4448 (1990), Ohagi et al., Nucl. Acids Res. 18:7159 (1990)). Schlessinger, PCT Publication WO 92/01050 (Jan. 23, 1992) disclosed human RPTP-xcex1, xcex2 and xcex3, and described the nature of the structural homologies among the conserved domains of these three RPTPs and other members of this protein family. The murine RPTP-xcex1 has 794 amino acids, whereas the human RPTP-xcex1 has 802 amino acids. RPTP-xcex1 has an intracellular domain homologous to the catalytic domains of other tyrosine phosphatases. The 142 amino acid extracellular domain (including signal peptide of RPTP-xcex1) has a high serine and threonine content (32%) and 8 potential N-glycosylation sites. cDNA clones have been produced that code for the RPTP-xcex1, and RPTP-xcex1 has been expressed from eukaryotic hosts. Northern analysis was used to identify the natural expression of RPTP-xcex1 in various cells and tissues. A polyclonal antibody to RPTP-xcex1, produced by immunization with a synthetic peptide of RPTP-xcex1, identifies a 130 kDa protein in cells transfected with a cDNA clone encoding a portion of RPTP-xcex1.
Another RPTP, HePTP (Jirik et al., FASEB J. 4:82082 (1990) Abstract 2253) was discovered by screening a cDNA library derived from a hepatoblastoma cell line, HepG2, with a probe encoding the two PTP domains of LCA. The HePTP gene appeared to be expressed in a variety of human and murine cell lines and tissues.
Since the initial purification, sequencing, and cloning of a PTP, additional potential PTPs have been identified at a rapid pace. The number of different PTPs that have been identified is increasing steadily, leading to speculations that this family may be as large as the PTK family (Hunter (1989) supra).
Conserved amino acid sequences designated xe2x80x9cconsensus sequencesxe2x80x9d have been identified in the catalytic domains of known PTPS (Krueger et al., EMBO J. 9:3241-3252 (1990) and Yi et al., Mol. Cell. Biol. 12:836-846 (1992), which are incorporated herein by reference). Yi et al. aligned the catalytic phosphatase domain sequences of the following PTPs: LCA, PTP1B, TCPTP, LAR, DLAR, and HPTPxcex1, HPTPxcex2, and HPTPxcex3. This alignment includes the following xe2x80x9cconsensus sequencesxe2x80x9d (Yi et al., supra, FIG. 2(A)):
1. K C X X Y W P [SEQ ID NO:1]
2. H C S X G X G R X G [SEQ ID NO:2]
Krueger et al., aligned the catalytic phosphatase domain sequences of PTP1B, TCPTP, LAR, LCA, HPTPxcex1, xcex2, 8, xcex5 and xcex6, and DLAR and DPTP. This alignment includes the following xe2x80x9cconsensus sequences: (Krueger et al., supra, FIG. 7):
1. K C X X Y W P [SEQ ID NO:1]
2. H C S X G X G R X G [SEQ ID NO:2]
It is becoming clear that dephosphorylation of tyrosine residues can by itself function as an important regulatory mechanism. Dephosphorylation of a C-terminal tyrosine residue has been shown to activate tyrosine kinase activity in the src family of tyrosine kinases (Hunter, T. Cell 49:1-4 (1987)). Tyrosine dephosphorylation has been suggested to be an obligatory step in the mitotic activation of the maturation-promoting factor (MPF) kinase (Morla et al., Cell 58:193-203 (1989)). These observations point out the need in the art for understanding the mechanisms that regulate tyrosine phosphatase activity.
It is clear that further analysis of structure-function relationships among PTPs are needed to gain important understanding of the mechanisms of signal transduction, cell cycle progression and cell growth, and neoplastic transformation. Such understanding will also provide useful agents for regulating these processes and for treating diseases associated with their dysregulation.
The inventors describe herein the identification of a novel PTP, termed PTP S31. This novel PTP differs significantly in structure from previously reported PTPs. Further, several variants of this PTP have been identified. The present invention thus provides a PTP-S31 protein or glycoprotein which is a PTP or contains structural features known to be found in PTPs, as well as variants thereof.
When a PTP-S31 protein or glycoprotein of the present invention is one which occurs in nature, it is substantially free of other proteins or glycoproteins with which it is natively associated. A substantially pure PTP-S31 protein or glycoprotein of the invention may be produced by biochemical purification, by chemical means or by recombinant means in a prokaryotic or eukaryotic host, and is provided substantially free of other proteins with which it is natively associated. The PTP-S31 may have modified amino acids.
The invention is further directed to:
(1) a fragment of a PTP-S31 protein or glycoprotein;
(2) a PTP-S31 protein or glycoprotein having additional amino acids;
(3) a PTP-S31 protein or glycoprotein having substituted amino acids; and
(4) a PTP-S31 protein or glycoprotein having any combination of deleted, additional, or substituted amino acids.
In all cases the modified PTP-S31 protein or glycoprotein, or fragment thereof, possesses the desired biological activity.
The invention is further directed to a nucleic acid molecule comprising a nucleotide sequence encoding a PTP-S31 protein according to the invention. The nucleic acid molecule may be cDNA, genomic DNA or RNA. The invention is further directed to a nucleic acid construct in the form of an expression vehicle. Also provided are prokaryotic and eukaryotic host cells containing the expression vehicle.
Also included in the present invention is a process for preparing a PTP-S31 protein or glycoprotein of this invention, comprising:
(a) culturing a host capable of expressing a PTP-S31 protein or glycoprotein under culturing conditions,
(b) expressing the PTP-S31 protein or glycoprotein; and
(c) recovering the PTP-S31 protein or glycoprotein from the culture.
The invention is also directed to a polyclonal, monoclonal or chimeric antibody specific for a PTP-S31 protein or glycoprotein or for an epitope of a PTP-S31 protein or glycoprotein.
The invention is further directed to a method for detecting the presence, or measuring the quantity, of a PTP-S31 protein or glycoprotein in a sample, preferably a cell or a biological sample from a subject, comprising:
(a) contacting the sample, such as a preparation of cells or an extract thereof, with an antibody specific for an epitope of a PTP-S31 protein or glycoprotein; and
(b) detecting the binding of the antibody to sample material, or measuring the quantity of antibody bound,
thereby detecting the presence or measuring the quantity of the PTP-S31 protein or glycoprotein.
The invention is also directed to a method for detecting the presence of a nucleic acid encoding a normal or mutant PTP-S31 protein or glycoprotein in a sample, preferably a cell or biological sample of a subject, comprising:
(a) contacting the sample, such as a cell or an extract thereof, with an oligonucleotide probe encoding at least a portion of a normal or mutant PTP-S31 protein or glycoprotein under hybridizing conditions; and
(b) measuring the hybridization of the probe to nucleic acid of the cell,
thereby detecting the presence of the nucleic acid. The nucleic acid of the sample can be selectively amplified, for example, by using the polymerase chain reaction, prior to assay.
The present invention is also directed to a method for identifying or isolating in a sample, preferably a chemical or biological sample, a compound capable of binding to a PTP-S31 protein or glycoprotein, the method comprising:
(a) attaching a PTP-S31 protein or glycoprotein or the compound-binding portion thereof to a solid phase matrix or carrier;
(b) contacting the sample with the PTP-S31 bound to the solid phase matrix, allowing any compound to bind to said PTP-S31, and washing away any unbound material;
(c) detecting the presence of the compound bound to the solid phase.
For purposes of isolation, the bound compound is subjected to the additional step of (d) eluting the bound compound, thereby isolating the compound.
The invention includes a method for identifying an agent molecule capable of stimulating or inhibiting the enzymatic activity of PTP-S31, comprising:
(a) contacting the agent with a PTP-S31 protein or glycoprotein, or a fragment thereof, which PTP-S31 may be in pure form, in a membrane preparation, or in a whole live or fixed cell;
(b) incubating the mixture of step (a) for a sufficient interval;
(c) measuring the enzymatic activity of the PTP-S31;
(d) comparing the enzymatic activity to that of the PTP-S31 protein or glycoprotein incubated without the agent,
thereby determining whether the agent stimulates or inhibits the enzymatic activity.
In addition, the present invention provides methods for identifying agonists or antagonists of PTP-S31 action based on the ability of such agents to modulate interactions between (a) PTP-S31 and its target molecules or (b) PTP-S31 and molecules which regulate its enzymatic activity. Compounds identified by such methods may be useful to treat ,diseases associated with PTP-S31 dysfunction or with disordered signal transduction.